The objective
of this project was to obtain a source of diamine oxidase
(DAO) necessary for large-scale production of a simple
and rapid dipstick method for determining histamine in
processed and fresh seafood products. Human diamine oxidase
cDNA was inserted into two different vectors designed
to secrete the enzyme into the media of cultured insect
(Drosophila) cells. Neither expression of cloned
DAO from insect cells, nor optimized purification procedures
from pig kidneys, have to date provided sufficient quantities
of enzyme. The investigators were able to obtain the human
diamine oxidase cDNA and subclone it into two expression
vectors. However, expression of the diamine oxidase cDNA
proved much more difficult than anticipated. The investigators
obtained no discernable expression at all in their insect
cells. One problem might be the existence of a single
nucleotide mutation or deletion somewhere in the clone.
The investigators believe that a likely reason for their
inability to detect DAO in transiently transfected cells
is that the transfection was inefficient and too few cells
were producing enzyme. The investigators will continue
attempts to express the human DAO in insect cells since
they believe this is the best solution to the expression
problem.